The use of a human papillomavirus 18 promoter for tissue-specific expression in cervical carcinoma cells

ArticleinCellular & Molecular Biology Letters 16(3):477-92 · September 2011with12 Reads
DOI: 10.2478/s11658-011-0018-8 · Source: PubMed
The use of tissue-specific promoter elements in the treatment of cervical cancer has been explored in this paper. The P(105) promoter of human papillomavirus 18 (HPV18) was utilised to direct tissue-specific expression in a number of cell types. Expression was examined in three cervical carcinoma cell lines: HeLa (HPV18 positive), SiHa (HPV16 positive), and C33A cells (HPV negative); the epithelial cell line, H1299; and the foetal fibroblast cell line, MRC5, utilising a luciferase expression vector. Expression was highest in the cervical cell lines by a factor of at least 80. The effect of a number of mutations in the P(105) promoter on expression levels was examined. Three deletion constructs of the long control region (LCR) were investigated: an 800 bp fragment (LCR800), a 400 bp fragment (LCR400), and a 200 bp fragment (LCR200), as well as the full length product LCR of HPV18 (LCR1000). The LCR800 construct of the HPV18 P(105) promoter had the highest level of expression in the cervical cell lines and was also highest in the HPV18-positive HeLa cell line. Site-directed mutagenesis was then employed on the LCR800 construct to create four further constructs that each had inactivating mutations in one of the four E2 binding sites (E2BSs). Overall, this study indicated that the LCR800 construct of the HPV18 P(105) promoter could be utilised as a tissuerestricted promoter in cervical cancer cells.
    • "These experiments identified the location of a transcriptional enhancer, i.e. a piece of DNA with numerous regulatory elements that " hyperactivate " transcription . The activity is cell type dependent, and strong in many types of epithelial cells, for example, in HeLa cells, but weak in fibroblasts (Cripe et al., 1987; Gloss et al., 1987; Swift et al., 1987; Lung et al., 2011; Weyn et al., 2011). This property has been called an " epithelial-specific " or " keratinocyte-dependent " enhancer. "
    [Show abstract] [Hide abstract] ABSTRACT: The small DNA genomes of papillomaviruses contain a surprisingly large number of regulatory or cis-responsive elements, which regulate replication and transcription of the virus, and control details like specificity for certain epithelial cells, specificity for layers in squamous epithelia, feedback mechanisms and coupling between host cell physiology and virus biology. Most of these elements occur in the long control region, while others are located elsewhere in the genome. Many papillomaviruses show a similar composition of cis-responsive elements, although these are scattered and do not occur as long segments of sequence similarity. This review summarizes our knowledge of the regulatory elements in several well-studied Alphapapillomavirus types, and indicates some similarities to other papillomavirus genera, whose properties are yet poorly understood.
    Article · May 2013
  • [Show abstract] [Hide abstract] ABSTRACT: In this study, the effect of mutations in transcription factor-binding elements was investigated in the human papillomavirus (HPV) 18 P(105) promoter. Site-directed mutagenesis activities, in the AP1/YY1-, KRF-1-, GRE/YY1-, Sp1- and the double mutation (AP1/YY1- and GRE/YY1)-binding sites were assessed in five human cell lines: HeLa (HPV18-positive cervical carcinoma), SiHa (HPV16-positive cervical carcinoma), C33A (HPV-negative cervical carcinoma), H1299 (non-small cell lung carcinoma) and MRC-5 (foetal lung fibroblast). The results indicated that the GRE/YY1 mutation increased the HPV18 P(105) promoter activity in the cervical cell lines by 53-135%. In HeLa and SiHa cells, mutations in the AP1/YY1, KRF-1 and Sp1 transcription factor-binding sites resulted in reduced promoter activity. For C33A, mutations in KRF-1 and Sp1 reduced the promoter activity, while the GRE/YY1 mutation increased the activity. The double mutation, AP1/YY1 and GRE/YY1, appeared to display an additive effect of the two individual mutations in cervical cells. Compared with HeLa cells, HPV18 P(105) promoter activity was more than 80-fold lower in H1299 cells and more than 500-fold lower in MRC-5 cells. Hence in this study, a comprehensive site-directed mutagenesis analysis, of important transcription factor-binding elements, in the HPV18 P(105) promoter was accomplished in a range of human cell lines. In particular, we concluded that HPV-induced factors were extremely important in the transcriptional activity of the HPV18 P(105) promoter.
    Article · Feb 2012